Airway epithelial cells are highly differentiated to perform critical functions for mucosal immunity and mucociliary clearance within the airway lumen. The unique morphology of ciliated and non-ciliated airway epithelial cells is characterized by a specialized set of receptors, channels, and anti-microbial proteins that must be localized in a polarized fashion to the apical or basolateral membranes. It is known that failure of differentiation or localization of apical proteins is the pathologic basis of lung disease in cystic fibrosis and other diseases. However, molecular pathways directing cell polarity are not well understood in airway epithelial cells. To further understand the regulation of epithelial cell differentiation, we previously cloned and initially characterized the forkhead transcription factor Foxj1 (HFH-4). We found that Foxj1 expression is restricted to ciliated epithelial cells, is temporally related to ciliogenesis, and that transgenic mice deficient in Foxj1 lack cilia. This proposal is based on a more detailed analysis of the Foxj1-/- mouse indicating the function of Foxj1 is broader than regulation of ciliogenesis and includes organization of the apical membrane of airway epithelial cells through regulation of cell membrane-cytoskeletal linker proteins. This hypothesis is based on our observations that Foxj1-/- epithelial cells have a failure of apical localization of (1) ciliary basal bodies, (2) transmembrane ion channels, epidermal growth factor receptor, and B2 adrenergic receptor, (3) and importantly, apical membrane-cytoskeletal linker protein ezrin and associated PDZ-domain protein EBP5O. In contrast, basolateral proteins are normally localized. Thus, in the absence of Foxj1 there is a failure of the apical membrane scaffolding proteins to localize and establish apical airway epithelial cell organization required for epithelial cell function and maintenance of the airway mileu. Accordingly, we propose biochemical and genetic approaches for in vivo analysis of the following specific aims. First, we will characterize the relationship between Foxj1 expression and apical membrane cytoskeletal proteins by evaluation of expression, localization, and function of apical ezrin, and ezrin-associated cytoskeletal proteins. Second, we will determine the role of Foxj1 in regulation of the apical compartment by assessment of apical membrane-cytoskeletal linker protein activation in Foxj1 deficient cells, characterize the effect of ezrin disruption on the phenotype of airway epithelial cells, and evaluate role of intracellular trafficking genes to regulate the Foxj1 null phenotype. Completion of these aims will provide important information regarding proteins required for airway epithelial cell differentiation and maintenance of lung function.